CN102586284A - Transcription factor derived from trichoderma reesei and coding gene and application thereof - Google Patents
Transcription factor derived from trichoderma reesei and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a transcription factor derived from trichoderma reesei and a coding gene and application thereof. The transcription factor complementary deoxyribose nucleic acid (cDNA) is obtained through separation from the trichoderma reesei, a nucleotide sequence is indicated as SEQ ID No.1, and an amino acid sequence of coded protein is indicated as SEQ ID No.2. The invention further provides expression vectors and recombination host cells of the transcription factor cDNA. The invention further provides use of the transcription factor cDNA in regulating growth and development and stress resistance of the trichoderma reesei.
Description
Technical field
The present invention relates to a kind of and fungi growth and grow relevant transcription factor cDNA and proteins encoded thereof; Relate in particular to from fungi Trichodermareesei (Trichoderma reesei) institute the transcription factor TrTF1 cDNA sequence and the encoded protein thereof of separating, cloning; The invention still further relates to the expression vector that contains this cDNA sequence and they the regulation and control Trichodermareesei grow and resistance in purposes, the invention belongs to the separation and the Application Areas thereof of the transcription factor that comes from Trichodermareesei.
Background technology
Trichodermareesei (Trichoderma reesei) is a kind of natural warm saprophytic fungus of having a liking for that extensively exists, and at first from cotton duck, separates (Mandels et al., 1957) during the World War II.Trichodermareesei can be secreted and produce a lot of enzymes; Comprise cellulase, hemicellulase and proteolytic enzyme and glycase; Especially cellulase content is very high; The excretory cellulase has been widely used in papermaking for many years, (Miettinen-Oinonen et al., 2002 in the industries such as wine brewing and food; Boer et al., 2002; Cherry et al., 2003).In recent years, along with the aggravation of energy dilemma, the research of the sustainable renewable energy resources becomes current research focus.Utilize reproducible cellulose resource to produce bio-ethanol (bioethanol) and become one of effective way that overcomes energy dilemma.In the flow process of producing bio-ethanol, it is a vital step of whole process that the cellulase hydrolysis Mierocrystalline cellulose produces glucose.Therefore, Trichodermareesei is as a kind of industrial bacterium that is widely used in the production of cellulose enzyme, and how the scientific research personnel improves and done number of research projects aspect the cellulase activity both at home and abroad.Find in the research, only can not solve the problem of cellulase activity fully through optimization to the transformation of bacterial strain and fermenting process.Want to settle the matter once and for all, the whole growth that must fundamentally understand Trichodermareesei is grown and the process of the inducing of cellulase, expression and the whole regulation and control of excretory.
In recent years; The progress of Trichodermareesei molecular biology aspect very rapidly; Especially in the announcement of Trichodermareesei genome sequence in 2008, through to genome analysis, prediction, for more deep understanding Trichodermareesei whole growth growth course has been established better basis.
As a kind of industrial strain of widespread use, the correlative study on the Trichodermareesei molecular level concentrates on the aspects such as abduction delivering of cellulase mostly, separately from the relevant report of the angle research Trichodermareesei that grows seldom.The gene that participates in the cellulase pathways metabolism of at present having cloned and having analyzed has as follows: ACE1, ACE2, XYR1, CRE1, HAP2/3/5 complex body, XYL1, GAL1, GNA1, GNA3, (Ilm é n et al., 1996 such as PGL1; Aro et al., 2001; Aro et al., 2002; Mach-Aigner et al., 2008; Stricker et al., 2006; Stricker et al., 2008; Seibel et al., 2009; Schmoll et al., 2009; Lim ó n et al., 2011 etc.).This shows; The emphasis of research all is partial to directly or the gene of indirect participation cellulase pathways metabolism; But the metabolic regulation of cellulase is far above the adjusting that receives these genes, and it receives the approach of other many unknowns in the Trichodermareesei whole growth growth course and assists completion.
The expression of known trichoderma reesei cellulase is mainly regulated and control on transcriptional level.Found that 5 transcriptional regulators participate in this process, XYR1 wherein, ACE2, the HAP2/3/5 complex body plays positive regulating and controlling effect, and ACE1 and CRE1 play the negative regulation effect.Meanwhile, these genes all also affect the others of Trichodermareesei growth course.Therefore, find and find new transcription factor to have great significance.
Identify and clone new transcription factor, especially those genes direct or indirect and the cellulase regulation and control can provide certain reference for utilizing the molecule means to transform Trichodermareesei raising cellulase yield.At present; The Li's Trichoderma strains through the molecular biology method transformation directly is not used for large-scale commercial prodn; Therefore; Utilize the Protocols in Molecular Biology clone and identify that new transcription factor has very important meaning to the metabolism network of overall understanding Trichodermareesei, also lays the foundation for final Protocols in Molecular Biology is applied to industrial production.
Summary of the invention
One of the object of the invention provides a kind of that from Trichodermareesei (Trichoderma reesei), separate, clone and its grow relevant transcription factor cDNA and proteins encoded thereof.
Two of the object of the invention provides expression vector that contains above-mentioned transcription factor cDNA and the recombinant host cell that contains this expression vector.
Three of the object of the invention is that said transcription factor cDNA and proteins encoded thereof are applied to growing or its resistance of Trichodermareesei (Trichoderma reesei).
Be to realize above-mentioned purpose, one aspect of the present invention provide a kind of from Trichodermareesei (Trichodermareesei) isolating TrTF1 transcription factor cDNA, its polynucleotide be (a) and (b) or (c) shown in:
(a), the polynucleotide shown in the SEQ ID No.1;
(b), amino acid whose polynucleotide shown in the coding SEQ ID No.2;
(c), with the polynucleotide that the complementary sequence of SEQ ID NO:1 can be hybridized in rigorous hybridization conditions, the coded proteinic amino acid of this Nucleotide is shown in the SEQ ID NO:2.
Preferably, of the present invention from Trichodermareesei isolating transcription factor cDNA be the polynucleotide sequence shown in the SEQ IDNo.1.
In addition, the present invention also provide in the Trichodermareesei (Trichoderma reesei) the full gene of isolating TrTF1 transcription factor, its polynucleotide sequence is shown in the SEQ ID NO:3.
Another aspect of the present invention provides the TrTF1 transcription factor of being grown by the coded regulation and control Trichodermareesei of above-mentioned transcription factor cDNA, its amino acid be (a) or (b) shown in:
(a), the amino acid shown in the SEQ ID No.2;
(b), with the replacement of the amino acid shown in the SEQ ID No.2 through one or more amino-acid residues, disappearance or/and insert derive obtain still have functional transcription factor or an active protein variant shown in the SEQ ID No.2;
Preferably, transcription factor of the present invention is the amino acid shown in the SEQ ID No.2.
Described " a plurality of " mean 2-10 usually, are preferably 2-6, and more preferably 2-4, this depends on the position or the amino acid whose kind of amino-acid residue in the transcription factor three-dimensional structure; Described " replacement " is meant respectively and replaces one or more amino-acid residues with the different amino acid residue; Described " disappearance " is meant the minimizing of amino-acid residue quantity, that is to say to lack one or more amino-acid residue respectively; Described " insertion " is meant the change of amino acid residue sequence, relative natural molecule, and said change causes adding one or more amino-acid residues.
Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these working method are generally this area and understand.For example, can prepare the aminoacid sequence variant or the fragment of transcription factor through the sudden change of DNA, the method for wherein said mutagenesis or change polynucleotide is the convention of this area institute.Conservative replacement is that a kind of amino-acid residue is replaced to the another kind of amino acid with similar quality.
Transcription factor of the present invention and encoding sox thereof comprise naturally occurring sequence and two kinds of forms of variant." variant " means similar basically sequence, and for polynucleotide, variant comprises the disappearance, insertion of the one or more Nucleotide of one or more site in the natural polynucleotide or/and replacement.For polynucleotide, conservative variant comprises those variants that do not change amino acid sequence coded owing to the degeneracy of genetic code.Naturally occurring variant like that can be identified through existing Protocols in Molecular Biology.The variant polynucleotide also comprise the polynucleotide in synthetic source, the amino acid whose polynucleotide variant shown in the SEQ ID No.2 of still encoding that for example adopts site-directed mutagenesis or obtain through the method for recombinating.
Among the present invention isolating TrTF1 transcription factor gene cause declines of Trichodermareesei sporulation quantity, conidiophore metamorphosis after inserting inactivation, resist the ability drop of ambient pressure environment.It is thus clear that; The present invention isolating TrTF1 transcription factor gene and Trichodermareesei grow and degeneration-resistant susceptibility relevant; Thus; Of the present inventionly provided on the one hand a kind of method that Trichodermareesei grows or improves Trichodermareesei resistance of regulating and control again, having comprised: the isolating transcription factor cDNA of the present invention is incorporated in the Trichodermareesei induced transcription factor cDNA expression.
The present invention further provides expression vector that contains said transcription factor gene and the host cell that contains this expression vector.
Be connected with expression regulation element transcription factor cDNA according to the invention is exercisable, obtain in Trichodermareesei, to express this expression carrier." exercisable connection " refers to functional connection between two or more elements, and the element of exercisable connection can be adjacency or non-adjacent.This expression vector can be by 5 ' end non-coding region, and the Nucleotide shown in the SEQ ID No.1 and 3 ' non-coding region are formed, and wherein, described 5 ' end non-coding region can comprise that promoter sequence, enhancer sequence are or/and the translation enhancement sequences; Described 3 ' non-coding region can comprise terminator sequence, mRNA cutting sequence etc.In addition, those skilled in the art can be optimized the Nucleotide shown in the SEQ ID No.1 to strengthen the expression in Trichodermareesei.For example.Can adopt the preference codon of Trichodermareesei to be optimized synthetic polyribonucleotides to strengthen the expression in Trichodermareesei.
Further, can with the present invention isolating TrTF1 transcription factor cDNA polynucleotide sequence be applied to seek that other receives the regulatory gene of TrTF1 in the Trichodermareesei as probe;
In addition; Can also the coded albumen of TrTF1 transcription factor cDNA of the present invention be that target protein is searched for the albumen that has identity function in other fungi in ncbi database; As: sickle-like bacteria (Fusarium oxysporum); Gibberella saubinetii (Gibberella zeae), aspergillus oryzae (Aspergillus oryzae), black mold (Aspergillus niger) etc.
The term definition that arrives involved in the present invention
Only if in addition definition, otherwise all technology used herein and scientific terminology all have with those skilled in the art and understand identical implication usually.Though in practice of the present invention or test, can use any method, device and the material similar or equivalent, describe preferred method, device and material now with person described herein.
Term " rigorous hybridization conditions " means known LIS and pyritous condition in affiliated field.Usually, under rigorous condition, but but probe is higher (for example above at least 2 times of backgrounds with the detection level of other sequence hybridization with the detection level ratio of its target sequence hybridization.Rigorous hybridization conditions is a sequence dependent, will different, long sequence specific hybrids under comparatively high temps under different environmental conditions.Preciseness or wash conditions through control hybridization can be identified and probe 100% complementary target sequence.Detailed guidance for nucleic acid hybridization can be with reference to relevant document (Tijssen; Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategy of nucleic acid assays.1993).More specifically, said rigorous condition is selected as usually and is lower than the heat fusion joint (T of distinguished sequence under regulation ionic strength pH
m) about 5-10 ℃.T
mFor under equilibrium state 50% with target complementary probe hybridization during to target sequence residing temperature (under specify ion intensity, pH and nucleic acid concentration) (because of the excessive existence of target sequence, so at T
m50% probe is occupied under equilibrium state down).Rigorous condition can be following condition: wherein be lower than about 1.0M Na ion concentration in 7.0 to 8.3 times salt concn of pH; Be generally about 0.01 to 1.0M Na ion concentration (or other salt); And temperature is at least about 30 ℃ for short probe (including, but is not limited to 10 to 50 Nucleotide), and for long probe (including, but is not limited to greater than 50 Nucleotide), is at least about 60 ℃.Rigorous condition also can realize such as the destabilizing agent of methane amide through adding.For selectivity or specific hybrid, positive signal can be the background hybridization of twice at least, is 10 times of background hybridizations according to circumstances.The rigorous hybridization conditions of exemplary can be following: 50% methane amide, and 5 * SSC and 1%SDS cultivate down at 42 ℃; Or 5 * SSC, 1%SDS cultivates down at 65 ℃, in 0.2 * SSC, washs and under 65 ℃, in 0.1%SDS, washs.Said washing can be carried out 5,15,30,60,120 minutes or the longer time.
Term " recombinant host cell " or " host cell " mean the cell that comprises polynucleotide of the present invention; And no matter use which kind of method to insert, for example directly absorb to produce recombinant host cell, known other method in transduction, f pairing or the affiliated field.Exogenous polynucleotide for example can remain, and the nonconformity carrier of plasmid perhaps can be integrated in the host genome.
Term " polynucleotide " means deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Only if specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in said term, said analogue have the binding characteristic that is similar to reference nucleic acid and carry out metabolism with the mode of the Nucleotide that is similar to natural generation.Only if other specific limited, otherwise said term also means oligonucleotide analogs, it comprises PNA (PNAG3), used DNA analogue (thiophosphatephosphorothioate, phosphoramide acid esters or the like) in antisense technology.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and clear and definite specified sequence.Specific; Can realize that through mixing base and/or the substituted sequence of Hypoxanthine deoxyriboside residue degenerate codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) through producing one of them or selected more than one (or all) codon the 3rd; People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); People such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " polypeptide ", " peptide " and " albumen " exchange in this article and use to mean the polymkeric substance of amino-acid residue.That is, be equally applicable to describe peptide and describe albumen and vice versa to the description of polypeptide.Said term is applicable to natural generation aminoacid polymers and one of them or the aminoacid polymers that above amino-acid residue is a non-naturally encoded amino acids.As used herein, the amino acid chain of any length contained in said term, and it comprises full-length proteins (being antigen), and wherein amino-acid residue connects via the covalency peptide bond.
Description of drawings
Fig. 1 is used for T-DNA and inserts the vector construction synoptic diagram.
Fig. 2 T-DNA on position synoptic diagram.
Fig. 3 Trichodermareesei wild-type QM9414 (A and C) and insertion two mutants ATMT-39 (B and D) colonial morphology figure; Wherein, A and B are picture on the PDA substratum, and C and D are picture on the MM substratum.
Trichodermareesei wild-type QM9414 (A) under Fig. 4 ordinary optical microscope and insertion two mutants ATMT-39 (B) mycelia aspect graph;
Fig. 5 Trichodermareesei wild-type QM9414 (A) and insertion two mutants ATMT-39 (B) mycelia form sem photograph.
Fig. 6 Trichodermareesei wild-type QM9414 and insertion two mutants ATMT-39 growth velocity comparison diagram under differing temps.
Fig. 7 Trichodermareesei wild-type QM9414 and insertion two mutants ATMT-39 growth velocity comparison diagram under different concns NaCl condition.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Sequence according to Totomycin in the pCB1003 carrier (Carroll et al.Fungal Genet.Newslett. (1994) 41:22.); The design primer; Pcr amplification Totomycin fragment is inserted into pCAMBIA1300 carrier (Chen et al.Appl.Environ.Microbiol. (2000) 66 (10): be built into pCAMBIA1300-1 4510-4513) with this fragment then.Concrete grammar is following: at first, adopts high-fidelity PCR method, utilizes the pCB1003 plasmid as template, and amplification Totomycin fragment, upstream and downstream is introduced XhoI site (italic is represented restriction enzyme site) simultaneously in the design of primers, so that next step experimental implementation.
hphp1：5’-
TATTGAAGGAGGATTTTTGGGC-3’
hphp2：5’-
GCTCTTGTTCGGTCGGCAFCTA-3’
The PCR system is: template pCB1003 plasmid 1 μ l, and Taq plus polysaccharase 1 μ l, each 0.4 μ l of primer (100mM), Taq plus damping fluid 5 μ l, dNTP (25mM) 1 μ l, all the other waters complement to 50 μ l.
The PCR reaction conditions is: 94 ℃ 5 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 1 minute 30 seconds) amount to 35 circulations, 72 ℃ 10 minutes.
Be connected to behind the PCR product purification on the pUCM-T carrier, cut with the XhoI enzyme then, be connected to after cutting on the pCAMBIA1300 carrier of cutting with same XhoI enzyme, promptly build the pCAMBIA1300-1 carrier.Building process is seen Fig. 1; The restriction enzyme site of writing a Chinese character in simplified form among Fig. 1 is respectively: Xh:XhoI, E:EcoRI, Sa:SacI, K:KpnI, S:SmaI, B:BamHI, X:XbaI, P:PstI, H:HindIII.LB (left border) is a left arm, and RB (right border) is a right arm.
Embodiment 2ATMT transforms the pCAMBIA1300-1 carrier to Trichodermareesei
The main freeze-thaw method that adopts transforms, and the pCAMBIA1300-1 carrier is transferred among the Agrobacterium AGL1.Select the single colony inoculation of an Agrobacterium from the LB flat board (containing 50 μ g/ml kantlex) of fresh culture and contain the LB of kantlex 28 ℃ of 200rpm incubated overnight in 5ml; 200-400 μ l nutrient solution was transferred to 5ml in second day and contain inducing among the liquid culture medium A IM of 200 μ mol/L AS and 50 μ g/ml Kna, about 0.15, the 28 ℃ of cultivation 5-6h of OD value makes OD
600Reach 0.5-0.6.
The collection of Trichodermareesei spore: wash spore with the 5ml sterile purified water from the PDA flat board of cultivating 10d, in the test tube of sterile glass beads is housed, after the vortex vibration; After three layers of lens wiping paper filter; 5000rpm is centrifugal, after twice of sterile water wash, counts with blood counting chamber; 100 μ l cultured Agrobacterium AGL-1 bacterium liquid and the good Trichodermareesei spore suspension of 100 μ l dilution are mixed, and evenly coating mixed liquid is cultivated 48h for 20 ℃ altogether on the AIM flat board that is covered with nitrocellulose filter (NC) then; Film is cut into small pieces, and 25 ℃ of cultivations occur up to transformant on selective medium PDA.Contain 200 μ g/ml Totomycin, 400 μ g/ml cephamycins (cefotaxime), 60 μ g/ml Streptomycin sulphates in the selective medium.After transformant grows, screening once more on the PDA flat board that contains 200 μ g/ml Totomycin again.The transformant that grows is all chosen to the PDA flat board, observed colonial morphology.
1.ATMT-39 two mutants inserts confirming of site: from two mutants ATMT-39, extract genomic dna,, carry out the NEST pcr amplification, obtain to confirm on position behind the insertion sequencing fragment as template.NEST primer sequence that PCR uses is according to T-DNA sequences Design on the pCAMBIA1300 carrier, and sequence is following:
LB4：5’-GCGTTACCCAACTTAATCGC-3’
RB4：5’-AGCGCAACGCAATTAATGTG-3’
LB5：5’-GCCAGGGTTTTCCCAGTCAC-3’
RB5：5’-AGTTAGCTCACTCATTAGGC-3’
LB6：5’-CGTTGTAAAACGACGGCCAG-3’
RB6：5’-ACCCCAGGCTTTACACTTTA-3’
After QM9414 genome after extracting cut with the EcoRI enzyme, use T
4Ligase enzyme connects, and 4 ℃ of connections are carried out follow-up PCR reaction to connect product as template then.Linked system is: the genome enzyme is cut product 4 μ l, T
4Ligase enzyme 1 μ l, T
4Ligase enzyme damping fluid 1 μ l, all the other use ddH
2O supplies 10 μ l.
NEST pcr amplification step and PCR reaction conditions, system: PCR is that template is carried out pcr amplification with the DNA that connects certainly of purifying for the first time, and primer adopts LB4/RB4; PCR uses PCR product dilution for the first time to be template for 1000 times for the second time, and primer adopts LB5/RB5, and PCR uses PCR product dilution for the second time to be template for 1000 times for the third time, and primer adopts LB6/RB6, obtains the T-DNA flanking sequence through above-mentioned a series of pcr amplifications.The PCR reaction conditions is: 94 ℃ of preparatory sex change 45s, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 4min, 35 circulations; After last loop ends, extend below 10min in 72 ℃.The PCR reaction system is: 0.5 μ lLATaq (5U/ μ l), and 10 * PCR reaction buffer, 5 μ l, each 0.4 μ l of primer (100mM), dNTP (25mM) 1 μ l, template 10-100ng, all the other waters complement to 50 μ l.
To the resulting PCR product of pcr amplification for the third time; Glue reclaims the back order-checking; Resulting sequencing result is compared in Trichodermareesei genomic data (http://genome.jgi-psf.org/Trire2/Trire2.home.html) and ncbi database, confirms the on position (Fig. 2) of T-DNA.
2.TrTF1 the clone of genomic dna: according to measured sequence, seek corresponding gene at the Trichodermareesei genome database, according to information in the genome database, design primer, amplification TrTF1 genomic dna.Primer sequence is following:
39p1：5’-AAGGATCCACCCCCGTACTCCCTATCTCCTGT-3’
39p2：5’-AAGGATCCGTGCGGCCCTGTACCATTTTC-3’
The PCR system is: template QM9414 genomic dna 1 μ l, and Taq plus polysaccharase 1 μ l, each 0.4 μ l of primer (100mM), Taq plus damping fluid 5 μ l, dNTP (25mM) 1 μ l, all the other waters complement to 50 μ l.
The PCR reaction conditions is: 94 ℃ 5 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 1 minute 30 seconds) amount to 35 circulations, 72 ℃ 10 minutes.
Be connected on the pUCM-T carrier after gained PCR product glue reclaims and order-checking, sequence is SEQ IDNO:3.
The ligation condition: PCR glue reclaims product 3 μ l, T
4Ligase enzyme 1 μ l, T
4Ligase enzyme damping fluid 1 μ l, pUCM-T carrier 1 μ l, all the other use ddH
2O supplies 10 μ l.
3.TrTF1cDNA the clone: adopt the TRIZOL method to extract RNA among the QM9414, reverse transcription obtains cDNA, and pcr amplification obtains the cDNA sequence.
The TRIZOL method is extracted total RNA:
It is extremely Powdered with liquid nitrogen grinding to collect about 1g left and right sides Trichodermareesei mycelia, adds 2ml Trizol, and room temperature is placed 5min, makes its abundant cracking.The centrifugal 5min of 8500rpm abandons deposition; Press 200ul chloroform/ml Trizol and add chloroform, vibration mixing 15 seconds, room temperature is placed 3min.4 ℃ of centrifugal 15min of 8500rpm.Draw the upper strata water to another centrifuge tube; Press 0.5ml Virahol/ml Trizol and add the Virahol mixing, room temperature is placed 10min, and 4 ℃ of centrifugal 10min of 8500rpm abandon supernatant, and RNA is sunken to the pipe end.Add 75% ethanol by 1ml 75% ethanol/ml Trizol, gentle vibration centrifuge tube, deposition suspends.4 ℃ of centrifugal 5min of 6500rpm abandon supernatant as far as possible.Dry 10min on ice.Use an amount of H
2O (about 100ul) dissolving RNA sample is surveyed the quantitative RNA concentration of O.D value.
CDNA is synthetic:
Adopt the synthetic cDNA of First Strand cDNA Synthesis Kit test kit.With the total RNA of 1 μ g, 1 μ l oligo (dT), 18 primers add in the pipe of an aseptic free nucleic acid pollution, add free nucleic acid ddH
2O is settled to 100 μ l, and cooled on ice behind the mixing if RNA template GC content is high or comprise secondary structure, needs to place on ice behind 65 ℃ of temperature bath 5min again.Add 5 * reaction buffer, 4 μ l then, dNTP Mix (10mM) 2 μ l, M-MuLV Reverse Transcriptase (20u/ μ l) 2 μ l, RiboLock
TMRNase Inhibitor (20u/ μ l) 1 μ l makes that the total reaction system is 20 μ l; The back 37 ℃ of temperature of soft mixing are bathed 60min, if RNA template GC content is high, can temperature of reaction be brought up to 45 ℃; Bathe the 5min termination reaction 70 ℃ of temperature at last.
The cDNA sequence amplification of TrTF1:
Primer sequence is following:
39cDNAp1：5’-ATGCCTACGAAAAGGTGAAGGA-3’
39cDNAp2：5’-CGGCTCTGCATCAACCCGACC-3’
The PCR system is: template cDNA 1 μ l, and Taq plus polysaccharase 1 μ l, each 0.4 μ l of primer (100mM), Taq plus damping fluid 5 μ l, dNTP (25mM) 1 μ l, all the other waters complement to 50 μ l.
The PCR reaction conditions is: 94 ℃ 5 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 1 minute 30 seconds) amount to 35 circulations, 72 ℃ 10 minutes.
Be connected on the pUCM-T carrier after gained PCR product glue reclaims and order-checking.Sequence is SEQ IDNO:1.
The ligation condition: PCR glue reclaims product 3 μ l, T
4Ligase enzyme 1 μ l, T
4Ligase enzyme damping fluid 1 μ l, pUCM-T carrier 1 μ l, all the other use ddH
2O supplies 10 μ l.
4.TrTF1 protein function prediction
The TrTF1 protein sequence is predicted on http://www.ebi.ac.uk/.
The mensuration of embodiment 4ATMT-39 colonial morphology, spore shape, sporulation quantity and growth velocity
1. colonial morphology:
ATMT-39 two mutants and QM9414 are connected to respectively on PDA and the MM flat board, cultivate after 3 days for 30 ℃ and take colonial morphology (Fig. 3).Wherein, the PDA culture medium prescription is: potato 200g in every liter of substratum, glucose 20g, agar 20g; The MM culture medium prescription is: glucose 20g in every liter of substratum, (NH
4)
2SO
45g, KH
2PO
415g, MgSO
40.6g, CaCl
20.6g, FeSO
40.005g, MnSO
40.016g, ZnSO
40.0014g, CoCl
20.002g, pH5.5.
2. conidiophore and conidium form:
Observation under the opticmicroscope is that the flat board of cultivating 3 days is used sterile water wash, with spreading rod the mycelia and the spore of planar surface is washed off, and the agar that will have the living mycelia of base then is cut into the fritter that size is about 1 * 0.5cm, is positioned on the slide glass; After slide glass is positioned in the 9cm flat board (keep dull and stereotyped in certain humidity), take out behind the 16h, observe mycelia, conidiophore and conidium form (Fig. 4) down as for opticmicroscope.
The plate edge of cultivating 48h is cut into the fritter that size is about 1 * 0.5cm with blade, and on the ESEM Stage microscope, the back is with the above back of liquid nitrogen freeze-drying 30min metal spraying, scanning electron microscopic observation (Fig. 5).
3. the mensuration of sporulation quantity and growth velocity:
ATMT-39 two mutants and QM9414 are connected on the PDA flat board, cultivate after 3 days for 30 ℃ and measure colony diameter, calculate growth velocity.The PDA flat board of cultivating 3 days is made a call to 3 holes with punch tool, and the position is respectively near the bacterium colony center, bacterium colony mid-way and colony edge; The bacterium piece of accomplishing fluently is placed the 10ml centrifuge tube, add 5 granulated glass spherees in the centrifuge tube simultaneously; Concussion with the blood counting chamber counting, was measured the result and is seen table 1 after 5 minutes on the vortex oscillation device.
Table 1
The degeneration-resistant sensitivity testing of embodiment 5ATMT-39
Be primarily aimed at different concns NaCl, different concns H
2O
2And growth velocity is measured the variation of degeneration-resistant susceptibility under the differing temps.
Differing temps is to the influence of degeneration-resistant susceptibility: ATMT-39 and QM9414 are cultivated down respectively at 30 ℃ and 37 ℃ of culture condition, measure colony diameter every day, up to the 3rd day (Fig. 6).
Different concns NaCl is to the influence of degeneration-resistant susceptibility: ATMT-39 and QM9414 is connected to respectively contains 0.2M, and 0.4M, 0.8M, on the PDA flat board of 1.0M different concns NaCl, colony diameter was measured in 30 ℃ of cultivations every day, up to the 5th day (Fig. 7).
Different concns H
2O
2Influence to degeneration-resistant susceptibility: ATMT-39 and QM9414 be connected to respectively contain 1mM, 3mM, the different concns H of 5mM
2O
2The PDA flat board on, colony diameter is measured in 30 ℃ of cultivations every day, up to the 5th day, measures the result and sees table 2.
Table 2
Claims (10)
1. isolating transcription factor cDNA from Trichodermareesei (Trichoderma reesei), its polynucleotide be (a) and (b) or (c) shown in:
(a), the polynucleotide shown in the SEQ ID No.1;
(b), amino acid whose polynucleotide shown in the coding SEQ ID No.2;
(c), with the polynucleotide that the complementary sequence of SEQ ID NO:1 can be hybridized in rigorous hybridization conditions, the coded proteinic amino acid of these polynucleotide is shown in the SEQ ID NO:2.
2. isolating transcription factor gene from Trichodermareesei (Trichoderma reesei), its polynucleotide sequence is shown in the SEQ ID NO:3.
3. the coded albumen of the said transcription factor cDNA of claim 1.
4. the coded albumen of the said transcription factor gene of claim 2.
5. according to claim 3 or 4 described albumen, it is characterized in that, its amino acid be (a) or (b) shown in:
(a), the amino acid shown in the SEQ ID No.2;
(b), with the replacement of the amino acid shown in the SEQ ID No.2 through one or more amino-acid residues, disappearance or/and insert derive obtain still have functional transcription factor or an active protein variant shown in the SEQ ID No.2.
6. the expression vector that contains the said transcription factor cDNA of claim 1.
7. the expression vector that contains the said transcription factor gene of claim 2.
8. the recombinant host cell that contains claim 6 or 7 said expression vectors.
9. said transcription factor cDNA of claim 1 or the said transcription factor gene of claim 2 are in the purposes of regulation and control Trichodermareesei in growing.
10. said transcription factor cDNA of claim 1 or the said transcription factor gene of claim 2 are in regulation and control or improve the purposes in the Trichodermareesei resistance.
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| CN 201210082203 CN102586284B (en) | 2011-12-15 | 2012-03-23 | Transcription factor derived from trichoderma reesei and coding gene and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070609A (en) * | 2017-12-27 | 2018-05-25 | 福建师范大学 | By the use of trichoderma reesei as the method for host expresses recombinant protein |
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Non-Patent Citations (4)
| Title |
|---|
| XIAO-HONG LIU ET AL.: "An autophagy gene, TrATG5, affects conidiospore differentiation in Trichoderma reesei.", 《RESEARCH IN MICROBIOLOGY》 * |
| 凌敏等: "康氏木霉中调控纤维素酶形成的两个调控因子的克隆及功能性分析", 《生物工程学报》 * |
| 李辉等: "里氏木霉产纤维素酶的诱导和合成机理研究进展", 《中国酿造》 * |
| 赫荣琳: "TrATG5基因对里氏木霉生长发育的影响", 《中国博士学位论文全文数据库基础科学辑 》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070609A (en) * | 2017-12-27 | 2018-05-25 | 福建师范大学 | By the use of trichoderma reesei as the method for host expresses recombinant protein |
| CN108070609B (en) * | 2017-12-27 | 2020-12-18 | 福建师范大学 | Method for expressing recombinant protein by using trichoderma reesei as host |
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| CN102586284B (en) | 2013-08-28 |
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